Cells possess multiple G protein signaling pathways that contribute to many different cellular responses, and the relative participation of these individual pathways in cell development, function, survival and disease is actively being investigated. However, there is a lack of suitable drugs which selectively interfere with specific G protein signaling events, and which can be applied to the study of intact cells and the treatment of disease. With the exception of receptor-derived peptides (Freissmuth, et al. (1999) Trends Pharmacol. Sci. 20:237-245; Kuliopulos & Covic (2003) Life Sci. 74:255-262; Covic, et al. (2002) Nat. Med. 8:1161-1165), and G protein α subunit C-terminal peptides (Freissmuth, et al. (1999) supra; D'Ursi, et al. (2006) Mol. Pharmacol. 69:727-36; Mazzoni, et al. (2000) Mol. Pharmacol. 58:226-236), which modulate receptor-G protein coupling, the field has for the most part been limited to drug candidates that interfere with different downstream kinases or other downstream effectors. However, the separate G protein pathways share many of these downstream targets, thereby making it challenging to modulate a specific cellular function associated with a certain G protein.
Switch I and II regions of Gα subunits are structurally and functionally analogous to those first described in the small GTPase Ras (Pai, et al. (1990) EMBO J. 8:2351-2359), while switch III region is unique to heterotrimeric G proteins. These switch regions are domains which undergo conformational changes during G protein activation induced by GDP-GTP exchange (Pai, et al. (1990) supra; Milburn, et al. (1990) Science 247:939-945; Tong, et al. (1991) J. Mol. Biol. 217:503-516; Sondek, et al. (1994) Nature 372:276-279; Lambright, et al. (1994) Nature 369:621-628; Coleman & Sprang (1998) Biochemistry 37:14376-14385). Furthermore, crystallization studies have revealed that the switch I region (SR1) contains critical sites for not only binding GTP, but also other subunits (Lambright, et al. (1994) supra). Consequently, this region is thought to be important for modulating activation of the α-subunit, as well as for regulating the affinity of the α-subunit for the β-subunits (Lambright, et al. (1994) supra). Other studies have established that the Gα13 subunit is critical for G13-mediated Rho activation (Gratacap, et al. (2001) J. Biol. Chem. 276:47906-47913; Ponimaskin, et al. (2000) FEBS Lett. 478:173-177; Kuner, et al. (2002) Eur. J. Neurosci. 16:2333-2341; Chen, et al. (2001) Nat. Struct. Biol. 8:805-809; Wells, et al. (2001) J. Biol. Chem. 276:28897-28905; Wells, et al. (2002) J. Biol. Chem. 277:1174-1181) presumably through its interaction with p115RhoGEF (Wells, et al. (2001) supra; Meigs, et al. (2005) J. Biol. Chem. 280:18049-18055; Nakamura, et al. (2004) Mol. Pharmacol. 66:1029-1034). Indeed, it has been found that Gα13 binds both the RGS and DH domains of p115RhoGEF, and that this Gα13 binding to p115RhoGEF increased its GTPase activity (Wells, et al. (2001) supra). Furthermore, the direct participation of SR1 and SR2 in this p115RhoGEF binding interaction was provided by studies (Meigs, et al. (2005) supra) demonstrating that replacement of the 196 to 203 sequence of Gα12 SR1 (or the replacement of the 244 to 249 sequence of Gα12 SR2) by the sequence Asn-Ala-Ala-Ile-Arg-Ser (SEQ ID NO:1) reduced Gα12 binding to p115RhoGEF in vitro. In addition, it has been demonstrated that a single mutation, i.e., K204A of G13 switch I region, impairs the regulatory function of Gα13 on p115 and LARG RhoGEF in vitro activities (Nakamura, et al. (2004) supra). A p115RhoGEF-RGS-G13 crystal structure model has also been described in which both SR1 and SR2 of Gα13 are important for binding interactions with p115RhoGEF (Chen, et al. (2001) supra). Taken together, these results suggest that Gα switch regions are important for G protein downstream effector activation.